Immunogenical complex formed by vaccinal antigens encapsulated by nanostructured mesoporous silica

ABSTRACT

The present invention relates to a product named “immunogenical complex”, which comprises an adjuvant characterized by solid particles of highly ordinated nanostructured mesoporous silica, preferably, SBA-15 Silica, and vaccinal antigens of several natures, encapsulated in the referred to adjuvants. The immunogenical complex of the present invention allows the presentation of the antigens that compose it to lymphocytes, in a safe, gradual and extended way, which leads to a more efficient immunological memory, increases the immunogenicity of the antigen and improves the production of antibodies. This ensures an efficient immunological protection with fewer amounts of antigens and/or less repetitions of vaccinal doses. In addition, the characteristics of the immunogenical complex of the present invention promotes effective immunity induction, homogeneous in “god and bad respondent” individuals.

The present invention relates to the immunology field.

The present invention relates to a product designated “immunogeniccomplex”, effective in increasing immunogenicity, constituted byvaccinal antigens encapsulated by solid particles of highly orderednanostructured mesoporous silica acting as adjuvant, as shown in thepresent invention. The encapsulation by mesoporous silicas protects theantigens from degradation by macrophages and extends its exposure tolymphocytes, promoting improved immune response effective for inductionof antibody production, either in high or in low responder individuals.The immunogenic complex of the present invention may bring benefit forthe general immunological activity to antigens of distinct types:biologically active peptides, toxins, viral and bacterial vaccines.

The immune response of human beings to vaccinal antigens varies due toparticular factors. Several individuals vaccinated with the sameantigen, under the same conditions, produce responses that vary inintensity and duration. Such variation is a determinant factor of theintensity and duration of vaccines protective effect.

After standardized antigenic stimulation, the individuals that replyproducing protective titers of antibodies are named high responders andthose that do not produce protective titers are the low or evennon-responders.

The development of safe and effective strategies for improvement of theimmune response, either from high or from low responders is of utmostinterest. In the first case, through production of protective responseswith lower amount of antigen, or long-lasting response withoutre-exposure to the antigen. In the second case, through production aprotective response with stimuli that, otherwise, would be insufficient.

Currently, this problem is only partially solved by the use of adjuvantsthat are defined as materials that extend the specific immune responseof the organism to certain antigen [Edelman, R.; Tacket, C. O.;Adjuvants Intern. Ver. Immunol, 7 (1990) 51], modifying the form throughwhich the epitopes (antigenic determinants) are presented to cells ofthe immune system or raising the immunogenicity thereof. Othercharacteristics desirable for an adjuvant are: to sustain the stimulusperiod, increase the presentation time of the antigen and delay thecatabolism thereof.

Apparently, many adjuvants exercise their activity by toxic actionsagainst the macrophages. There are also adjuvants that modulate theimmune response to certain antigen as, for example, inducing thepredominant expression of an immunoglobulin isotype, for example an IgG.[Hadjipetrou-Kourounakis, L.; Möller, E.; Scand. J. Immunol., 19 (1984)219].

The adjuvants licensed and largely used in human vaccines are thederivatives of aluminum salts, such as aluminum hydroxide or phosphate.However, these do not induce an immunological response substantiallyhigh and long lasting or qualitatively selective in relation to thedesirable subclass of IgG antibodies and to the cytokines involved.

There are other adjuvants used in veterinary such as the IncompleteFreund Adjuvant [IFA] and the Complete Freund Adjuvant [CFA] thatpromote the undesirable formation of nodules, abscess or granulomes inthe local of administration. Other adjuvants are: Lipid A, Microspheresand Liposomes, none of which are destined for use in humans.

Thus, the interest in the development of safe and effective strategiesfor improvement of the immune response remains evident, either from highor low responders. In this way, the advancement in the sciences ofmaterials area is enabling the preparation of new compounds withimproved properties and potential application in several areas.

The inorganic porous solids present important industrial applications incatalytic and separation processes. These materials, due to thestructural and surface properties thereof, allow the access of moleculesto its nanostructures, thus increasing the catalytic and sorptionactivity thereof.

The porous materials currently used may be classified in three classesbased on its peculiar microstructure: paracrystalline amorphoussupports, materials with modified layers and crystalline molecularsieves. The differences in micro and mesostructure of these materialsare important, either for its sortive and catalytic behavior, as well asin the properties used for their characterization, such as: superficialarea, pores size and distribution thereof, the presence or absence ofX-ray diffraction standards (XRD) and the details in such standards, andthe aspect of the materials when its microstructure is studied bytransmission electronic microscopy (TEM) and electrons diffractionmethods.

Amorphous and paracrystalline materials represent an important class ofporous inorganic solids that have been used for many years in industrialapplications. Typical examples of these materials are the amorphoussilica, regularly used in formulation of catalysts, and the transitiveparacrystalline alumina, used as supports for acid solid catalysts andpetroleum modified catalysts. The term amorphous is used in this contextfor indicating a material that does not present a long-range order,although nearly all materials are ordered at a certain extent, at leastin local scale. An alternative term that is being used to describe thesematerials is: “Indifferent X-Ray”. The microstructure of silica consistsof particles of 10-25 nm of dense amorphous silica, with porosityresulting from empty spaces between particles. Since there is nolong-range order in these materials, the pore size tends to bedistributed within a wide range. This lack of order is also manifestedin the X-ray diffraction standard (XRD), which usually appears withoutthe characteristic peaks.

Paracrystalline materials, such as transitive alumina, have beenpresenting a wide distribution of the pores size, but well defined fromthe X-ray diffraction standard, that usually consists of some widebands. The microstructure of these materials consists of smallcrystalline regions of condensed alumina phases and the porosity of thematerials is the result of irregular empty spaces between these regions.Considering that in the case of one material or another, there is nolong-range order controlling the pores size in the material, thevariability in such sizes is typically very high. The pore size in thesematerials comprises a band named mesopores that ranges between 1.3 to 20nm.

In contrast with these solids, structurally little defined, are thematerials, which distribution of pore sizes is very narrow, since it iscontrolled from the crystalline nature of the materials, accuratelyrepeated, designated as microstructures. These materials are designatedas “molecular sieves”, and the most important examples are the zeolytes.

Such molecular sieves, natural or synthetic, include a wide variety ofcrystalline silicates containing positive ions.

In general, porous substances are divided by the pore size, for example,substances with pores size of less than 2 nm are classified asmicroporous, between 2 to 50 nm as mesoporous substances and over 50 nmare classified as macroporous substances.

A series of mesoporous molecular sieves, including MCM-41 and MCM-48,were described in U.S. Pat. Nos. 5,057,296 and 5,102,643. Thesemolecular sieves show a structure in which the mesopores, uniform insize, are regularly arranged. MCM-41 has a uniform structure showing ahexagonal arrangement of direct mesopores, such as honeycomb, and has aspecific surface area of 1000 m²/g obtained by BET method.

Molecular sieves have been produced using inorganic or organic cationsas mold. These mesoporous molecular sieves are synthesized through aliquid crystal mechanism using surfactants as molds and have theadvantage that the size of the pores may be adjusted in the range of 1.6to 10 nm, through the control of surfactant type or synthetic conditionsemployed during the production process.

Molecular sieves designated SBA-1, SBA-2 and SBA-3 were described inScience (1995) 268:1324. Its channels are regularly arranged, while theconstituent atoms show an arrangement similar to that of amorphoussilica. Mesoporous molecular sieves have regularly organized channels,larger than those existing in zeolytes, in this way capacitating itsapplication in adsorption, isolation or reactions of catalyticconversion of relatively large molecules.

U.S. Pat. No. 6,592,764 found a family of high quality mesoporoussilicas, hydrothermal stability and of ultra-extensive pores size,through the synthesis with the use of an amphiphilic block copolymer inacid medium. A member of the family, SBA-15, has highly orderedmesostructure, hexagonal in two dimensions (p6 mm) similar to ahoneycomb. Other structures as cubic in cage form, or three-dimensionalhexagonal are also formed. A calcination procedure at 500° C. yieldsporous structures with high BET surface area of 690 to 1040 m²/g, andpores volume above 2.5 cm³/g, large interplanary distances d(100) of7.45 to 45 nm, pores size of 4.6 to 50 nm and the thickness of silicawall of 3.1 to 6.4 nm. SBA-15 may be prepared with an extensive band ofpores size and thickness of pore wall at low temperature (35 to 80° C.),using a variety commercially available of biodegradable and non-toxicamphiphilic block copolymer, including tri-block polyoxyalkaline.

The unique properties of SBA-15 make it an attractive material forseveral applications, including bio-application, for example, fixing ofbiologically active species. However, no document reporting theinfluence of these materials on immune responsiveness was identified, onthe contrary, the literature would suggest its non-exploration for thispurpose.

Experiments concerning the influence of amorphous silica in the immuneresponse, specifically on macrophages, were already carried out,however, at that time they did not involve the role of silica asadjuvant [Allison, A. C.; Harington, J. S.; Birbeck, M.; J. Exp. Med.,124 (1966) 141; Kampschmidt, R. F.; Worthington, M. L.; Mesecher, M. I.;J. Leukocyte Biol., 39 (1986) 123; Lotzova, E.; Cudkowicz, G.; J.Immunol., 113 (1974) 798; Lotzova, E; Gallagher, M. T.; Trentin, J. J.Biomedicine, 22(5) 387 1975; Vogel, S. N.; English, K. E.; O'brien, A.D.; Infect. Immun., 38 (1982) 681].

In another experiment [Gennari, M.; Bolthillier, Y.; Ibanez, O. M.;Ferreira, V. C. A.; Mevel, J. C.; Reis, M. A.; Piatti, R. M.; Ribeiro,O. G.; Biozzi, G.; Ann. Inst. Pasteur Immunol., 138 (1987) 359.], thegenetically modified mice according to the low or high antibodyproduction were used, and in which the suspensions of colloidal silicawere administered during 4 consecutive days, prior to immunization withparticulated antigen, namely, heterologous erythrocytes. These studiesshowed that there is a significant increase in the production ofantibodies of low responder animals, and this improvement would bedirectly related with the silica action on macrophages, affecting someof its functions, changing the viability of these cells and leading thereduction of the antigen catabolism, thus favoring the presentation ofthe antigen to lymphocytes.

Thus, these effects were analyzed comparing the responses of mousestrains that express distinct characteristics in relation to thefunctionality of its macrophages. It was achieved using an experimentalmodel that selects the mice strains with the phenotypes of maximum orminimum response of antibodies. Such strains were obtained aftercrossbreeding between individuals with extreme phenotypes duringconsecutive generations. After about 15 generations, animals presentingextreme phenotypes for the level of antibodies achieved homozygosis ofthe relevant alleles controlling responsiveness against certain antigen.With this model it was possible to obtain the high [H] or low [L]antibody responder lines of Selection IVA [Cabrera, W. H.; Ibanez, O.M.; Oliveira, S. L.; Sant'Anna, O. A.; Siqueira, M.; Mouton, D.; Biozzi,G.; Immunogenetics, 16 (1982) 583]. The differences of responses inthese animals are related to the higher (L_(IVA) mice line) or lower(H_(IVA) mice line) macrophages catabolic activity, prejudicing orfavoring, respectively, the effective presentation of antigens.

The above-mentioned studies showed that when L_(IVA) mice are previouslyand extensively treated with amorphous silica suspensions, and thenimmunized with an antigen, had its antibodies production increased,approaching to the responses of the H_(IVA) mice. On the other hand,[Biozzi, G.; Mouton, D.; Sant'Anna, O. A.; Passos, H. C.; Gennari, M.;Reis, M. H.; Ferreira, V. C. A.; Heumann, A. M.; Bouthillier, Y.;Ibanez, O. M.; Stiffel, C.; Siqueira, M.; Current Topics In MicrobiologyImmunology, 85 (1979) 31.], in another similar experimental model, inwhich H_(III) and L_(III) mice obtained by an independent geneticselection III were used, the modulation of antibody production of thelow responder mice was not observed, after treatment with the samesuspension of amorphous silica. It must be stressed that in theseH_(III) and L_(III) animals, the high or low levels of antibodiesproduction, does not correlate with the functionality of itsmacrophages, but to the potentiality of its lymphocytes.

These studies were fundamental to give support to understand the in vivorole of macrophages in immunization processes, in addition to showingthat for an efficient adjuvant used in the induction of immunity itshould protect the antigen administered against the highly catabolicactivity of macrophages and suitably present the antigenic determinantsto lymphocytes.

In large vaccine campaigns, uniform immunization products and processesare generally adopted for a large and heterogeneous group ofindividuals. Under these conditions, the production of variable titersof antibodies can be observed, some non-protective. It hinders theefficient immunization of part of the individuals.

Such fact is explained by the mechanisms shown in the above-mentionedexperiments and originates from the phenotype variability of theindividuals of the same specie, which may be interpreted by theefficient form or not of presentation of the epitope to the lymphocytes.

For example, individuals with lymphocytes effector activity that couldbe classified from normal to very high, or macrophages activity fromreduced to normal, have a tendency to react more promptly, in relationto the production of antibodies, since the probability of the antigen tobe identified more efficiently by the lymphocytes is great. These wouldbe the “high responder” individuals in a natural population.

On the contrary, individuals that present from normal to reducedlymphocytes activity, and very high macrophages activity have a tendencyto more rapid catabolize the antigen administered. It leads to a lowerexposure of the antigen to lymphocytes and to an ineffective immuneresponse. These would be the “low responder” individuals in a naturalpopulation. This situation favors a natural selection of more resistantpathogens.

It is necessary to develop more efficient vaccines that would favor andpromote the production of protective antibody titers, even inindividuals that are low responders to the current vaccine formulations.Therefore, it is important that this differentiated cellular behaviormust be taken into consideration in the selection of the adjuvant,seeking to minimize the influence of the differentiating factors.

The application of this concept does not exist yet, and we miss productsand/or vaccines produced in accordance thereof.

One objective of the present invention is to show that antigensincorporated or encapsulated in nanostructured mesoporous silica form ahighly effective immunogenic complex that is efficient in the inductionof an immune response and that such nanostructured mesoporous silicas donot affect the viability and phagocytic capacity of macrophages inculture.

The present invention relates to a new immunogenic complex constitutedby antigens of several natures, encapsulated by highly orderednanostructured mesoporous silicas that act as adjuvants, improving theinduction of immunity and the production of antibodies to antigens,distinct concerning its nature, structure and complexity.

The immunogenic complex according to the present invention relates tothe product resulting from the combination of an antigen and particlesof nanostructured mesoporous silicas in specific proportions.

The immunogenic complex of the present invention allows the effectiveimmunization of the individuals that are low responders to products andprocesses currently used. It originates from more safe and effectivepresentation of the antigen to the lymphocytes.

The immunogenic complex of the present invention is constituted by atleast one antigen, which is incorporated or encapsulated by theparticles of nanostructured mesoporous silicas. In addition toeffectively acting as immunization adjuvant, silica particles also serveas support or matrix for bioactive species, in this case, immunogens.

The antigens that may be used in the formation of the immunogeniccomplex of the present invention include biologically active peptides,toxins, and viral and bacterial vaccines.

Although a wide range of nanostructured mesoporous silica may be used asadjuvants in the preparation of the immunogenic complex of the presentinvention, preferably, the silica designated as SBA-15 is used.

The highly ordered nanostructured mesoporous silica, SBA-15, is composedof silicon oxide particles with regular cavities and uniform in sizebetween 2 to 50 nanometers. The antigen is set in these nanocavities forthe encapsulation thereof. At the same time, this protects thedegradation by macrophages and carries it to gradual and more efficientpresentation to the lymphocytes, increasing the efficacy of the immuneprocess.

Methods of preparation of SBA-15 silica and similar mesoporous materialsare described in scientific articles (Zhao et al., Science (1998)279:548; J. Am. Chem. Soc. (1998) 120:6024; Matos et al., Chem. Mater.(2001) 13:1726), and in U.S. Pat. No. 6,592,764.

The objective of the present invention is also to present anincorporation or encapsulation process of the antigen in nanostructuredmesoporous silica, for preparation of the immunogenic complex.

The encapsulation of the antigens on silica occurs, in general, by meansof a process that comprises a mixture of a solution previously preparedcontaining the antigen with a silica suspension, both diluted inphysiological solution with pH of 7.4. The weight proportion of theantigen in relation to silica may range from 1:5 to 1:50, preferably is1:25. This preferred proportion might be read as 1 μg of antigen to 25μg of silica. The preparations are preferably carried out at roomtemperature and maintained under occasional stirring until about twohours prior to inoculation time.

Another objective of the present invention is to present the use ofimmunogenic complex in preparation of vaccine compositions forprophylactic use.

Pharmaceutical compositions, containing the immunogenic complex of thepresent invention and a pharmaceutically acceptable carrier, diluent orexcipient, are appropriate for medical and veterinary use.

One advantage of the present invention consists in the use of theimmunogenic complex to promote the induction of identical immuneresponse in both high and low responder individuals with fewer amountsof antigens. This aspect has a relevant economic and social importanceto public health.

The antigen is the raw material with higher cost for production ofvaccines. The reduction of the necessary amount for induction ofefficient immune response may lead to a substantial reduction inproduction cost of many vaccines.

On the other hand, the production of larger amounts of doses with thesame antigen amount has implications that surpass its simple economicaspects. There are antigens which production speed is limited even inthe absence of economic limiting factors. During epidemics, theoptimization and maximization of the immunization potential of smallerquantities of antigens may be essential for saving millions of lives.

Another very important aspect of the present invention consists in theextension of the stimuli period, through the increase of the time forpresentation of the antigen. It results in the induction of moreefficient immunological memory, guaranteeing protection with lowernumber of doses. Several vaccines need the administration of 3 or moredoses and periodic reinforcement to induce efficient protection. Thesustained presentation of the antigen may cause the reduction in thenumber of revaccinations in some cases.

This possibility is of great impact in public health since there is alow adhesion of many parents to regular vaccination programs,vaccinating their children mainly during large campaigns published bythe media. The possibility of inducing protective immunity with lowernumber of doses would minimize the lack of adhesion problem, taking moreadvantage of the campaigns and efficiently immunizing millions ofchildren, without need for returning.

DESCRIPTION OF FIGURES

FIG. 1. Small angle X-Ray diffraction of SBA-15 silica (NC) (naturalcalcined) and SBA-15 (GC) (ground calcined).

FIG. 2. Isotherm of nitrogen adsorption at 77K and the correspondingpore size distribution (PSD) of calcined silica SBA-15.

FIG. 3. Images of Transmission Electron Microscopy (TEM) of calcinedsilica SBA-15.

FIG. 4. Determination of mouse antibodies of the IgG isotypeanti-Intimin 1β of Escherichia coli, when comparing the adjuvantproperty of SBA-15 with other adjuvants after administration by theoral, intraperitoneal and subcutaneous routes.

The following examples are described as an illustration and there is nointention to use it for limiting the scope of the present invention.

EXAMPLE 1 Preparation and Characterization of Silica SBA-15 Component ofthe Immunogenical Complex as Immunization Adjuvant

In a reactor, 4 g of tri-block copolymer Pluronic P123 was dispersed,with magnetic stirring at 40° C., in 28 g of deionized water and 122 gof 2 M HCl solution. Then, 8.6 g of TEOS are added for obtaining ahomogeneous solution under mechanical and magnetic stirring at 40° C.About 15 minutes, after the addition of TEOS, the formation of thejelly-like precipitate may be observed. The gel is maintained understirring at 40° C. for 24 hours and, then, transferred to a Teflon-linedautoclave and placed in a sterilizer at a controlled temperature of 100°C. for 2 days. Then the solid product is filtered off, washed withdeionized water and air dried at room temperature. Finally, thesynthesized sample is calcined under dry N₂ flow at a flow rate of 100mLmin⁻¹ at 540° C., using a heating rate of 1° C.min⁻¹. After heatingfor 5 hours at 540° C., the flow of nitrogen gas is changed to air,without interruption of the process, and calcination continue for 3hours more.

The ordered bidimensional structure of SBA-15, in the form of channelsin hexagonal symmetry, was evaluated by small angle X-Ray diffraction(SAXRD) and measures of N₂ adsorption (to define the structural andsurface properties, in relation to the content of polymer present in thepreparation of the material) and by transmission electronic microscopy(TEM). The results of the material characterization are resumed in Table1 and illustrated by FIGS. 1, 2 and 3 of the present invention. Suchcharacteristics are appropriate for considering the material as anexcellent matrix for several molecular hosts.

TABLE 1 Results of SBA-15 characterization Parameter Result Small angleX-Ray diffraction (SAXRD) 12.7 nm (127 Å) Specific surface area (a) 900m²/g Total pore volume 1.39 cm³/g Maximum pore size (w)* 11.6 nm (116 Å)Thickness of silica wall (b)** 1.1 nm (11 Å) *Obtained by the pore sizedistribution (PSD); **b = a − wFIG. 1 shows the results of small angle X-Ray diffraction (SAXRD)obtained for the SBA-15 sample of calcined hexagonal type, in naturalstate (NC) and ground (GC). The results evidence that the structure ofthe ordered mesoporous materials (diffraction peaks) does not changeafter grinding the powders in agate mortar. The analysis and indexationof peaks are made after removal of the non-structured spreadingbackground.FIG. 2 shows the isotherm of nitrogen adsorption for calcined silicaSBA-15, which presented a high degree of ordination, as can be deducedfrom the declivity in isotherm adsorption in the step of capillarycondensation.FIG. 3 shows the transmission electron microscopy (TEM), which was usedto characterize the structural order of calcined silica SBA-15, wherethe order of parallel channels, particular of such type of material, canbe observed.

EXAMPLE 2 Determination of the Adsorption Percentage of the ModelAntigen by SBA-15

Using bovine serum albumin [BSA] as antigen, mixtures were made withSBA-15 at different proportions and, then, determination of adsorptionpercentage of antigen by silica for each proportion was made. Accordingto the results presented in Table 2, the proportion of 1 μg of BSA to 25μg of SBA-15 showed high adsorption percentage of BSA by SBA-15.

TABLE 2 Determination of the best proportion for adsorption of BovineSerum Albumin [66 kDa] in Silica SBA-15. BSA:SBA-15 Adsorption % 1:527.5 1:10 65.5 1:25 91

However, it is important to mention that due to the diversity ofantigens that can compose the immunogenic complex of the presentinvention, the optimization of the proportion between the antigen andSBA-15 should be reconsidered in function of the complexity of theantigen.

EXAMPLE 3 Demonstration of SBA-15 Effects on Macrophages

Experiments in vitro showed that nanostructured silica SBA-15 does notaffect the viability neither interferes in the macrophages phagocyticcapacity originating from the medulla, maintained in culture for up to30 hours. To the contrary, indicates to potentialize the phagocytosisthrough these cells. Table 3 shows that the treatment or not with SBA-15does not substantially interfere in the phagocytosis process of yeastcells in Strains: genetically selected for a low response [L_(IVA)],genetically heterogeneous [SWISS], or isogenic [BALB/c].

TABLE 3 In vitro experiment with macrophages of different mice strainsINFECTED No. OF STRAIN Presence of yeasts YEASTS L_(IVA) 20 μg SBA-15 +yeast 2 h 68.2% 496 20 μg SBA-15 + yeast 17 h 61.8% 350 10 μg SBA-15 +yeast 2 h 78.9% 474 10 μg SBA-15 + yeast 17 h 65.2% 326 2.5 μg SBA-15 +yeast 2 h 79.5% 503 2.5 μg SBA-15 + yeast 17 h 67.8% 379 Yeast 2 h 53.9%217 Yeast 6 h 59.2% 230 Yeast 17 h 82.8% 472 Yeast 21 h 59.9% 224 Yeast30 h 50.2% 164 SWISS 20 μg SBA-15 + yeast 2 h 84.9% 591 10 μg SBA-15 +yeast 2 h 81.7% 528 10 μg SBA-15 + yeast 17 h 70.8% 325 2.5 μg SBA-15 +yeast 2 h 81.9% 468 2.5 μg SBA-15 + yeast 17 h 74.7% 448 Yeast 2 h 78.2%479 Yeast 6 h 73.3% 437 Yeast 17 h 54.1% 284 Yeast 21 h 56.2% 218 Yeast30 h 53.9% 195 BALB/c Yeast 2 h   82% 622 Yeast 6 h 76.8% 438 Yeast 21 h68.5% 424 Yeast 30 h 51.5% 209

EXAMPLE 4 Adjuvant Effect of the Immunogenic Complex (Antigen:SBA-15) onAnti-Intβ Antibodies and Anti-Poison Micrurus ibiboca when Compared withthe Adjuvants Regularly Used in Mice Strains

Groups of 4-5 mice genetically selected according to high production ofantibodies [H_(III) line], or to the low response [L_(IVA) line], andmice of isogenic line [genetically identical animals] BALB/c were testedin distinct experiments. The potential effect of SBA-15 was evaluatedwith the measurement and the comparison of response to the recombinantprotein β-intimine [Int1β] of 16.5 kDa of the bacteria Escherichia coli,adsorbed in SBA-15 [1:10 Int1β:SBA-15] or admixed to Incomplete FreundAdjuvant (IFA). The response to antibodies formation was also evaluatedfor the total venom of the Elapidae snake family, Micrurus ibibobocagenus, composed by at least 20 proteins with molecular weight rangingfrom 84 to 7 kDa, adsorbed in SBA-15 [1:10 Micru:SBA-15], comparing theresponse to this venom admixed in IFA. All these experiments werecarried out following immunizations by subcutaneous route. Datapresented in Tables 4 and 5 [mean±standard deviation (log₂)] confirmthat SBA-15 is as efficient as IFA, promoting high antibody titers andbeing efficient in the immunological memory induction.

TABLE 4 Anti-Int1β Titer [log₂] 15 days after immunization SBA-15 IFAMice strain N x ± σ n x ± σ L_(IVA) 4 11.3 ± 0.5 4 4.5 ± 0.5 H₁₁₁ 4 11.3± 0.4 3 13.3 ± 0.5  BALB/c 4  6.2 ± 3.2 5 9.8 ± 2.3

TABLE 5 Anti-Micrurus Titer [log₂] 14 days after immunization SBA-15 IFAMice strain n x ± σ n x ± σ L_(IVA) 4 8.1 ± 0.5 3 5.2 ± 0.3 BALB/c 4 9.2± 1.3 4 6.4 ± 0.8

In addition, SBA-15, contrary to what occurs upon administration of IFA,does not lead to the formation of an apparent granuloma and, the localinflammatory response is insipid and, when measured at 24-48 hours afterthe inoculation of the immunogen in SBA-15 by subcutaneous routepresents very reduced levels of monocytes and nuclear polymorphous.

There is no apparent change in the behavior and vitality of mice thatreceived SBA-15 relatively to the control animals and, followed for 11months, no morphological change is observed in treated animals.

EXAMPLE 5 The Adjuvant Effect of the Immunogenic Complex(Antigen:SBA-15) on Anti-Intβ Antibodies in Function of Time whenCompared with the Adjuvants Normally Used

In another series of assays, groups of BALB/c mice were immunized withInt1β (from Escherichia coli) in SBA-15, Al(OH)₃ by oral route, or Int1βin SBA-15, Al(OH)₃ and IFA by subcutaneous and intraperitoneal route.The anti-Int1β responses were followed during a long time. FIG. 4presents the responses to the protein Intimin 1β of Escherichia coliaccording to distinct immunization routes. Means and standard deviationsof isogenic Strain BALB/c mice, followed up to 199 days [d] during theprimary responses [PR], immunized with the known adjuvants Al(OH)₃,Incomplete Freund Adjuvant (IFA) and the original SBA-15 nanostructuredsilica. It can be noted that the antibody levels remained high duringthroughout the analyzed period, especially in the group that receivedthe antigen in SBA-15.

Altogether the results clearly show that SBA-15 is a non-immunogenic,non-toxic and efficient carrier promoting both high response toantibodies and efficient immunological memory.

Highly ordered nanostructured mesoporous silicas, illustrated in thepresent invention by SBA-15 silica, provide promising systems forvaccinal preparations or compositions.

The invention claimed is:
 1. An immunogenic complex consistingessentially of i) particles of ordered nanostructured mesoporous silica,having pores of 2 to 50 nm in diameter, and ii) at least one antigen,wherein the proportion of antigen to silica particles is 1:5 to 1:50 andthe antigen is encapsulated by the particles of mesoporous silica, whichact as an adjuvant for immunization.
 2. The immunogenic complexaccording to claim 1, wherein the at least one antigen is selected fromthe group consisting of proteins, biologically active peptides, toxins,viruses and bacteria.
 3. The immunogenic complex according to claim 2,wherein the antigen is a bacterial protein antigen or a viral proteinantigen.
 4. The immunogenic complex according to claim 1, wherein theordered nanostructured mesoporous silica is a SBA-15 mesoporous silica.5. The immunogenic complex according to claim 1, wherein the proportionof antigen to silica particles is 1:25.
 6. The immunogenic complex ofclaim 1, that provides prolonged presentation of antigen to lymphocytesand results in improved immunologic memory response.
 7. A vaccinecomprising the immunogenic complex of claim 1, and a pharmaceuticallyacceptable carrier, diluent or excipient.
 8. A method for producing animmunogenic complex comprising mixing particles of orderednanostructured mesoporous silica, having pores of 2 to 50 nm indiameter, with at least one antigen, wherein the antigen and silicaparticles are mixed in a ratio of 1:5 to 1:50, whereby the antigen isenbapsulated by the particles of mesoporous silica, which act as anadjuvant for immunization.
 9. The method according to claim 8, whereinthe at least one antigen is selected from the group consisting ofproteins, biologically active peptides, toxins, viruses and bacteria.10. The method according to claim 9, wherein the antigen is a bacterialprotein antigen or viral protein antigen.
 11. The method according toclaim 8, wherein the highly ordered nanostructured mesoporous silica isa SBA-15 mesoporous silica.
 12. The immunogenic complex according toclaim 1, in which the pores of the mesoporous silica are hexagonallyclose packed.